SNX2112, a synthetic heat shock protein 90 inhibitor, has potent antitumor activity against HER kinase-dependent cancers

Abstract

Purpose: The heat shock protein 90 (Hsp90) chaperone plays an important role in transformation by regulating the conformational maturation and stability of oncogenic kinases and transcription factors. Ansamycins, such as 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), inhibit Hsp90 function; induce the degradation of Hsp90 client proteins such as HER2, and have shown activity in early clinical trials. However, the utility of these drugs has been limited by their hepatotoxicity, poor solubility, and poorly tolerated formulations.

Experimental design: We determined the pharmacodynamic and antitumor properties of a novel, synthetic Hsp90 inhibitor, SNX-2112, in cell culture and xenograft models of HER kinase-dependent cancers.

Results: We show in a panel of tumor cell lines that SNX-2112 and its prodrug SNX-5542 are Hsp90 inhibitors with properties and potency similar to that of 17-AAG, including: degradation of HER2, mutant epidermal growth factor receptor, and other client proteins, inhibition of extracellular signal-regulated kinase and Akt activation, and induction of a Rb-dependent G(1) arrest with subsequent apoptosis. SNX-5542 can be administered to mice orally on a daily schedule. Following oral administration, SNX-5542 is rapidly converted to SNX-2112, which accumulates in tumors relative to normal tissues. A single dose of SNX-5542 causes HER2 degradation and inhibits its downstream signaling for up to 24 h, and daily dosing results in regression of HER2-dependent xenografts. SNX-5542 also shows greater activity than 17-AAG in a non-small cell lung cancer xenograft model expressing mutant EGFR.

Conclusions: These results suggest that Hsp90 inhibition with SNX-2112 (delivered as a prodrug) may represent a promising therapeutic strategy for tumors whose growth and survival is dependent on Hsp90 clients.

Figures

Fig. 1

SNX-2112 and 17-AAG induce Hsp90 client degradation, inhibit Erk and Akt activation, and induce apoptosis in HER2-overexpressing cells. A, kinetics of client protein degradation in BT-474 cells treated with 1 μmol/L SNX-2112 and 17-AAG showing that both drugs induce client protein degradation with similar kinetics. Immunoblots were done using 25 to 50 μg of cell lysate. B, SNX-2112 and 17-AAG degraded Hsp90 clients with similar potency. Cells were treated with SNX-2112 or 17-AAG for 24 h followed by collection and analysis.

Fig. 2

Effect of 17-AAG and SNX-2112 on growth and cell cycle in multiple cell lines. A, indicated cell lines were seeded in 96-well plates and treated with increasing concentrations of 17-AAG or SNX-2112. The mean number of viable cells is reported as the percentage of the untreated count subtracting day 0 along with SD. Curves are representative of three independent experiments. B, BT-474 and MDA-468 cells were treated with the indicated concentrations of 17-AAG or SNX-2112 in triplicate and collected after 48 h. Nuclei were isolated, stained with ethidium bromide, and analyzed for DNA content and thus cell cycle distribution by flow cytometry. Apoptosis is reported as a mean of the sub-G1 fraction.

Fig. 3

Effect of a single, nontoxic dose of SNX-5542 in vivo. BT474 tumor-bearing mice were sacrificed and tumors were collected at the indicated times after treatment by oral gavage with a single dose of 50 mg/kg SNX-5542. Immunoblotting was done on tumor lysates using the indicated antibodies. Comparable data were obtained with several other doses including 75, 100, and 150 mg/kg (data not shown).

Fig. 4

Treatment with SNX-5542 caused tumor regression in a HER2-overexpressing breast cancer model and inhibition of tumor growth in a mutant EGFR non – small cell lung cancer model. A, mice with established BT474 tumor xenografts (10 mice per group) were randomized to treatment with SNX-5542 or to the control group. Mice were treated with 50 mg/kg of SNX-5542 by oral gavage on a Monday to Friday schedule. Treatment was discontinued after 4 wk (day 35) and measurements were continued until day 73. Animals in the control group were sacrificed at day 49 due to large tumor size. B, mice with established H1650 tumor xenografts (five mice per group) were randomized to treatment with 17-AAG (100 mg/kg i.p. Monday-Wednesday-Friday), SNX-5542 50 mg/kg orally Monday to Friday, or to the control group. Treatment was continued until sacrifice on day 37.

Fig. 5

Biodistribution of SNX-2112 in mice with established BT474 xenografts after oral administration of SNX-5542. A, mice with established BT474 xenografts were treated with a single oral dose of 75 mg/kg SNX-5542 with the animals sacrificed at the indicated times. Sample tissue from various organs was removed and homogenized, and the levels of SNX-2112 and SNX-5542 were assayed by MS. In parallel, lysates were prepared from the tumor and analyzed by immunoblotting with antibodies to HER2, P-Akt (Ser-473), phosphorylated mitogen-activated protein kinase, cyclin D1, cleaved poly(ADP)ribose polymerase, and p85-phosphatidylinositol 3-kinase. B, sample lysates of tumor and liver were prepared from BT-474 xenografts using the BioPlex Lysis kit. These were incubated with antibodies to P-Akt or P-Erk1/2 covalently attached to fluorescent beads. Reported values are relative fluorescent signals as detected by a Bio-Rad reader.